E-type cyclins (cyclin E1 and E2) are components of the core cell cycle machinery in mammalian cells. In order to address the function of these proteins in normal proliferation and in oncogenesis, we recently generated cyclin E1-/-E2-/- mice. We found that cyclin E-deficient embryos die during gestation, due to placental abnormalities. We were able to overcome the lethality by providing cyclin E1-/-E2-/- embryos with wild-type placentas, using the tetraploid blastocyst complementation technique. This finding indicates that E-type cyclins are dispensable for the development of the embryo proper. We derived fibroblasts (MEFs) from cyclin E1-/-E2-/- embryos, and we cultured them in vitro. We found that mutant cells proliferated normally under conditions of continuous cell cycling, but they were unable to re-enter cell cycle from the quiescent, G0 state. Our molecular analyses revealed that E-cyclins are critically required for loading of MCM2 replicative helicase onto DNA replication origins during G0->S phase progression. We also found that cyclin E1-/-E2-/- MEFs are resistant to oncogenic transformation by various oncogene pairs. E-cyclins are believed to drive cell cycle progression by binding and activating its kinase partner, cyclin-dependent kinase CDK2. However, very surprisingly, work from the laboratory of our collaborator on this grant, Dr. Barbacid revealed that MEFs derived from CDK2-/- embryos proliferate normally and do not display abnormalities seen in cyclin E-deficient MEFs. Importantly, no cyclin E-associated kinase activity was detected in these CDK2-/-cells. These results raise a possibility that E-cyclins may play kinase-independent roles in cell cycle progression. Several key issues remain unresolved: (1) Are there any kinase-independent functions for E-cyclins in cell proliferation? (2) Are E-type cyclins also required for cell cycle re-entry in vivo? (3) Are mice deficient in E-cyclins less susceptible to malignant transformation in vivo? We will address these questions in the following Specific Aims: (1) To study the molecular basis of cyclin E function in cell cycle progression. (2) To study the consequences of cyclin E ablation in adult tissues. (3) To determine whether mice deficient in E-type cyclins show reduced susceptibility to oncogenic transformation in vivo.